Protective Effects of Clenbuterol against Dexamethasone-Induced Masseter Muscle Atrophy and Myosin Heavy Chain Transition.

Glucocorticoid has a direct catabolic effect on skeletal muscle, leading to muscle atrophy, but no effective pharmacotherapy is available. We reported that clenbuterol (CB) induced masseter muscle hypertrophy and slow-to-fast myosin heavy chain (MHC) isoform transition through direct muscle β2-adrenergic receptor stimulation. Thus, we hypothesized that CB would antagonize glucocorticoid (dexamethasone; DEX)-induced muscle atrophy and fast-to-slow MHC isoform transition.We examined the effect of CB on DEX-induced masseter muscle atrophy by measuring masseter muscle weight, fiber diameter, cross-sectional area, and myosin heavy chain (MHC) composition. To elucidate the mechanisms involved, we used immunoblotting to study the effects of CB on muscle hypertrophic signaling (insulin growth factor 1 (IGF1) expression, Akt/mammalian target of rapamycin (mTOR) pathway, and calcineurin pathway) and atrophic signaling (Akt/Forkhead box-O (FOXO) pathway and myostatin expression) in masseter muscle of rats treated with DEX and/or CB.Masseter muscle weight in the DEX-treated group was significantly lower than that in the Control group, as expected, but co-treatment with CB suppressed the DEX-induced masseter muscle atrophy, concomitantly with inhibition of fast-to-slow MHC isoforms transition. Activation of the Akt/mTOR pathway in masseter muscle of the DEX-treated group was significantly inhibited compared to that of the Control group, and CB suppressed this inhibition. DEX also suppressed expression of IGF1 (positive regulator of muscle growth), and CB attenuated this inhibition. Myostatin protein expression was unchanged. CB had no effect on activation of the Akt/FOXO pathway. These results indicate that CB antagonizes DEX-induced muscle atrophy and fast-to-slow MHC isoform transition via modulation of Akt/mTOR activity and IGF1 expression. CB might be a useful pharmacological agent for treatment of glucocorticoid-induced muscle atrophy

Phenotypic Characterization of LEA Rat: A New Rat Model of Nonobese Type 2 Diabetes

Animal models have provided important information for the genetics and pathophysiology of diabetes. Here we have established a novel, nonobese rat strain with spontaneous diabetes, Long-Evans Agouti (LEA) rat derived from Long-Evans (LE) strain. The incidence of diabetes in the males was 10% at 6 months of age and 86% at 14 months, while none of the females developed diabetes. The blood glucose level in LEA male rats was between 200 and 300 mg/dl at 120 min according to OGTT. The glucose intolerance in correspondence with the impairment of insulin secretion was observed in male rats, which was the main cause of diabetes in LEA rats. Histological examination revealed that the reduction of β-cell mass was caused by progressive fibrosis in pancreatic islets in age-dependent manner. The intracytoplasmic hyaline droplet accumulation and the disappearance of tubular epithelial cell layer associated with thickening of basement membrane were evident in renal proximal tubules. The body mass index and glycaemic response to exogenous insulin were comparable to those of control rats. The unique characteristics of LEA rat are a great advantage not only to analyze the progression of diabetes, but also to disclose the genes involved in type 2 diabetes mellitus

Vidarabine, an anti-herpes agent, improves Porphyromonas gingivalis lipopolysaccharide-induced cardiac dysfunction in mice

Abstract In this work, we examined the involvement of type 5 adenylyl cyclase (AC5) in cardiac dysfunction induced in mice given Porphyromonas gingivalis lipopolysaccharide (PG-LPS) at a dose equivalent to the circulating levels in periodontitis (PD) patients. Cardiac function was significantly decreased in mice given PG-LPS compared to the control, but treatment for 1 week with the AC5 inhibitor vidarabine ameliorated the dysfunction. Cardiac fibrosis and myocyte apoptosis were significantly increased in the PG-LPS group, but vidarabine blocked these changes. The PG-LPS-induced cardiac dysfunction was associated with activation of cyclic AMP/Ca2+-calmodulin-dependent protein kinase II signaling and increased phospholamban phosphorylation at threonine 17. These results suggest that pharmacological AC5 inhibition may be a promising approach to treat PD-associated cardiovascular disease

Masseter muscle hypertrophy after treatment with CB and/or DEX for 2 weeks.

<p><b>(A)</b> Masseter muscle mass weight of CB was significantly greater, and that of DEX was significantly smaller than that of the Control (**<i>P</i> < 0.01 vs. Control in each case). Importantly, the DEX-mediated inhibition was suppressed by co-treatment with CB (CB+DEX vs. DEX, ^##<i>P</i> < 0.01). <b>(B)</b> Representative images of HE staining of masseter muscle of rats in the Control, CB, DEX, and CB+DEX groups. <i>Scale bar</i>: 100 μm. <b>(C)</b> Fiber diameter of CB was significantly greater and that of DEX was significantly smaller than that of the Control (**<i>P</i> < 0.01 vs. Control in each case). DEX-mediated decrease was suppressed by co-treatment with CB (CB+DEX vs. DEX, ^#<i>P</i> < 0.05). <b>(D)</b> CSA of CB was significantly greater (**<i>P</i> < 0.01) and that of DEX was significantly smaller (*<i>P</i> < 0.05) than that of the Control. DEX-mediated inhibition was suppressed by the co-treatment of CB (^##<i>P</i> < 0.01 vs. DEX). Masseter mass, fiber diameter and CSA in the Control were taken as 100% in each determination.</p

Effects of CB and DEX on IGF1 expression, myostatin expression, and Akt phosphorylation in masseter muscle after treatment with CB and/or DEX for 2 weeks.

<p><b>(A)</b> IGF1 expression in masseter muscle of the CB group was greater than that of the Control (**<i>P</i> < 0.01). Conversely, IGF1 expression of the DEX group was smaller than that of the Control (*<i>P</i> < 0.05). The DEX-mediated inhibition of IGF1 was suppressed by co-treatment with CB (CB+DEX vs. DEX, ^#<i>P</i> < 0.05) <b>(B)</b> Expression of myostatin protein was similar in all four groups (<i>P</i> = NS vs. Control in each case). <b>(C)</b> Phosphorylation of Akt on serine 473 in the CB, DEX, and CB+DEX groups was significantly greater than that in the Control (*<i>P</i> < 0.05, **<i>P</i> < 0.01 vs. Control). <b>(D)</b> Representative immunoblotting results for IGF1, myostatin, and phosphorylated Akt, and total Akt. The amount of expression or phosphorylation level in the Control was taken as 100% in each determination. p-Akt, phosphorylated Akt at serine 473; t-Akt, total Akt, GAPDH; glyceraldehyde 3- phosphate dehydrogenase</p

Muscle mass (mg) of rats in Control, CB, DEX or CB+DEX group.

<p>* p < 0.05,</p><p>** p < 0.01, vs. Control, n = 3.</p><p>Muscle mass (mg) of rats in Control, CB, DEX or CB+DEX group.</p

(A-C) Phosphorylation of NFATc1 on serine 259, phosphorylation of NFATc3 on serine 265, and expression of modulatory calcineurin-interacting protein 1 were similar in all four groups (<i>P</i> = NS vs. Control in each case).

<p><b>(D)</b> Representative immunoblotting results for phosphorylated and total NFATc1 and NFATc3, together with expression of modulatory calcineurin-interacting protein 1. The amount of phosphorylation (NFATc1 and NFATc3) or expression (modulatory calcineurin-interacting protein 1) in the Control was taken as 100% in each determination. p-NFATc1; phosphorylated NFATc1 at serine 259; t-NFATc1; total NFATc1; p-NFATc3, phosphorylated NFATc3 at serine 265, t-NFATc3; total NFATc3, GAPDH; glyceraldehyde 3- phosphate dehydrogenase</p

Changes of body weight, daily consumption of food and water, and daily intake of CB and energy.

<p>(<b>A)</b> Body weight (BW: g) of CB was similar to the Control (<i>P</i> = NS vs. Control). On the other hand, BW of both the DEX and CB+DEX groups was significantly smaller than that in the Control (**<i>P</i> < 0.01 vs. Control in each case). <b>(B-C)</b> No significant difference in daily consumption of food <b>(B)</b> or water <b>(C)</b> was observed among the CB, DEX, CB+DEX, and the Control groups (<i>P</i> = NS vs. Control in each case).<b>(D-E)</b> No significant difference was observed in daily intake of CB per baseline BW between the CB and CB+DEX groups <b>(D)</b> or in energy intake among the CB, DEX, CB+DEX, and the Control groups <b>(E)</b> (<i>P</i> = NS vs. Control in each case). The values of BW, consumption of food, consumption of water, CB intake, and energy intake in the Control group were taken as 100% in each determination.</p

Schematic illustration of the proposed pathways involved in the protective effects of CB against DEX-induced muscle atrophy.

<p>CB as well as DEX induces phosphorylation of Akt on serine 476. Activation of the Akt/mTOR pathway was increased by CB, but decreased by DEX. Activation of the Akt/FOXO pathway was similarly increased by CB and DEX, indicating that another pathway that is independent of Akt/FOXO pathway may account for the augmented expression of atrogin-1 and MuRF1 in DEX-treated group. Solid black lines represent findings in this study and solid grey lines represent findings reported previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128263#pone.0128263.ref005" target="_blank">5</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128263#pone.0128263.ref013" target="_blank">13</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128263#pone.0128263.ref016" target="_blank">16</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128263#pone.0128263.ref048" target="_blank">48</a>]. IGF1; insulin growth factor 1, REDD1; regulated in development and DNA damage responses 1, PI3K; phosphoinositide 3-kinase, KLF15; Kruppe-like factor 15</p